This grant concerns the mechanism of mRNA degradation by the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus. During lytic HSV infections, the half lives of both host and viral mRNAs are determined largely by the Vhs activity. At early times, Vhs accelerates the degradation of cellular mRNAs, contributing to an overall decrease in host protein synthesis. Following the onset of viral transcription, Vhs accelerates the turnover of viral mRNAs belonging to all kinetic classes. In this role, it helps determine viral mRNA levels and facilitates the sequential expression of different classes of viral genes. In addition, recent studies suggest that Vhs plays a significant role in HSV pathogenesis. Although Vhs does not discriminate between mRNAs, it does exhibit two important kinds of selectivity. First, it shows a strong preference for mRNAs, as opposed to non-messenger RNAs. Second, within mRNAs, Vhs initiates cleavage at preferred sites. For some mRNAs, these preferred sites are near regions of translation initiation. A potential clue to the mechanism of this selectivity comes from our observation that Vhs interacts with the cellular translation initiation factors elF4H and elF4All. The proposed studies are divided into four specific aims. Aim 1 will purify the Vhs polypeptide from HSV infected cells and from a recombinant complex of Vhs and elF4H, and characterize it biochemically. Aim 2 will map the sites at which Vhs cleaves selected target mRNAs in vivo, and compare them to the sites cleaved by purified Vhs, a recombinant Vhs-elF4H complex, and by Vhs in rabbit reticulocyte lysates. Aim 3 will determine the domains and residues of Vhs required for binding elF4H and elF4AII, and determine whether Vhs mutations, which block binding to either translation factor, abrogate targeted Vhs cleavage of mRNAs at preferred sites in vivo and in vitro. Finally, Aim 4 will investigate the mechanism by which the Vhs nuclease is targeted to preferred cleavage sites in mRNAs. Experiments will determine whether targeting is dependent upon an mRNA 5' end, ribosome scanning, the RNA helicase activity of elF4A, or the initiation factor elF4G. Together these studies should provide significant insights into the mechanism of Vhs-induced mRNA decay.